The Partnership for High-Throughput Enabled Biology of the Mitochondrial Proteome, located in the Center for Eukaryotic Structural Genomics (CESG), has the goal of applying technologies developed by the Protein Structure Initiative (PSI) to problems of interest to the community of biologists and biochemists who investigate the role of mitochondria in human health and disease. The mitochondrial proteome contains many ORFs, or domains of ORFs, that meet the PSl's criterion of "uniqueness" (low sequence similarity to proteins of known structure), many ORFs of major biomedical importance currently unrepresented by three-dimensional structures, and many proteins that lack functional characterization. The Mitochondrial Protein Partnership (MPP) has solicited additional nominations from the scientific community for mitochondrial protein targets of biological/biomedical interest. Targets meeting the criteria of "uniqueness" and/or "biological/biomedical relevance" will be cloned into a vector that enables automated wheat germ cell-free producfion of small amounts of protein for analysis of solubility, molecular mass, aggregation/dispersity, and thermal stability. These trials will identify proteins amenable to scaled-up protein producfion by CESG's cell-free or E. co//cell-based platforms. The MPP will offer targets that meet these criteria to the PSI Centers for High-Throughput Structure Determination as clones or, provided that suitable procedures are developed as part of MPP functional studies, as Se-Met-labeled protein samples. Functional investigations to be carried out by the MPP will include identification of protein-protein and protein-small molecule Interactions, enzymatic analyses, mitochondrial import assays, and effects of protein overexpression/depletion on the bioenergetic capacity of live cells. In support of these studies, and to help spotlight additional targets for structure determination, high-throughput wheat-germ screening will be extended to the entire human and mouse mitochondrial proteomes. The MPP will make available to members of the larger biological community samples of purified protein or fluorescent-tagged proteins (or clones coding for these) for their own functional investigations. The MPP will interface with the PSI Network by providing full information to the PSI Knowledgebase and by transferring all clones and plasmids to the PSI Materials Repository. PUBLIC HEALTH RELEVANCE: The mammalian mitochondrial proteome consists of -1,200 proteins, including hundreds with no biological characterization. Mitochondrial dysfunction is at the heart of more thatn 50 human diseases ranging from neonatal fatalities to adult-onset neurodegeneration, and is a likely contributor to type II diabetes, cancer, metabolic syndrome, obesity, and the aging process. Thus, mitochondrial proteomes represent a ripe focus area for high-throughput assisted structure-function investigations by the PSI Network.